Antimicrobials from an epigenetics based fungal metabolite screening program

ABSTRACT

Novel antimicrobial compounds against drug targets such as Eskape pathogens,  Leishmania donovani, Mycobacterium tuberculosis, Clostridium difficile, Naegleria fowleri , and cancer are presented herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims priority to U.S.Nonprovisional application Ser. No. 15/675,328, entitled “Antimicrobialsfrom an Epigenetics Based Fungal Metabolite Screening Program”, filedAug. 11, 2017, which is a continuation in part of and claims priority toU.S. Nonprovisional application Ser. No. 15/220,777, entitled“Antimicrobials from an Epigenetics Based Fungal Metabolite ScreeningProgram”, filed Jul. 27, 2016, which is a nonprovisional of and claimspriority to U.S. Provisional Patent Application No. 62/197,233, entitled“Antimicrobials from an Epigenetics Based Fungal Metabolite ScreeningProgram”, filed Jul. 27, 2015, the entire contents of each of which isherein incorporated into this disclosure.

GOVERNMENTAL SUPPORT

This invention was made with Government support under Grant No. AI103715awarded by the National Institutes of Health (NIH) and Grant No.AI103673 awarded by the National Institute of Allergy and InfectiousDiseases (NIAID). The Government has certain rights in the invention.

FIELD OF INVENTION

This invention relates to antimicrobials. Specifically, the inventionprovides new antimicrobials that are effective against various drugtargets such as Eskape pathogens, Leishmania donovani, Mycobacteriumtuberculosis, Clostridium difficile, Naegleria fowleri, and cancer.

BACKGROUND OF THE INVENTION

Sedentary and microbial organisms in all environments—marine,terrestrial, and fresh water—must produce secondary metabolites withwhich they can interact with the world around them. Micro-environments,such as fresh water ponds, inner-city forests, or coastal estuaries, toname a few, may be home to countless organisms that must respond toincredibly localized stressors that make no two environments exactly thesame. New chemical structures are emerging all the time from countlessenvironmental sources, and as threats to human health evolve, it couldcertainly be argued that natural products research is the way forwards.

Thanks to genomic advancements, it is clear that micro-organismscultured in the lab routinely produce only a fraction of the secondarymetabolites that are coded for in their DNA. (Gross, H. Strategies toUnravel the Function of Orphan Biosynthesis Pathways: Recent Examplesand Future Prospects. Applied Microbiology and Biotechnology. 2007, pp267-277). This is accomplished by the regulation of transcription byenzymes activated and deactivated based on environmental factors. (Bok,J. W.; Keller, N. P. LaeA, a Regulator of Secondary Metabolism inAspergillus Spp. Eukaryot. Cell 2004, 3 (2), 527-535). In filamentousfungi, it is known that most secondary metabolite genes are clustered toallow for the most efficient regulation and these clusters can beactivated or deactivated by culture conditions, resulting in vastlydifferent metabolite production. (Bok, J. W.; Keller, N. P. LaeA, aRegulator of Secondary Metabolism in Aspergillus Spp. Eukaryot. Cell2004, 3 (2), 527-535; Keller, N. P.; Hohn, T. M. REVIEW MetabolicPathway Gene Clusters in Filamentous Fungi. Fungal Genet. Biol. 1997,21, 17-29).

Once isolated, a micro-organism of interest can be cultured in the labunder any number of easily accessible stressors that can changesecondary metabolite production. Culture variations can be as simple aschanging the shape of the culture vessel, or as complex as the additionof biological material from another microbe or host organism. In thisway, a single strain can produce a multitude of different compounds.(Gross, H. Strategies to Unravel the Function of Orphan BiosynthesisPathways: Recent Examples and Future Prospects. Applied Microbiology andBiotechnology. 2007, pp 267-277; Lim, F. Y.; Sanchez, J. F.; Wang, C. C.C.; Keller, N. P. Toward Awakening Cryptic Secondary Metabolite GeneClusters in Filamentous Fungi. Methods Enzymol. 2012, 517, 303-324;Brakhage, A. A.; Schroeckh, V. Fungal Secondary Metabolites—Strategiesto Activate Silent Gene Clusters. Fungal Genet. Biol. 2011, 48 (1),15-22; Bode, H. B.; Bethe, B.; Hifs, R.; Zeeck, A. Big Effects fromSmall Changes: Possible Ways to Explore Nature's Chemical Diversity.ChemBioChem 2002, 3 (7), 619-627; Scherlach, K.; Hertweck, C. TriggeringCryptic Natural Product Biosynthesis in Microorganisms. Org. Biomol.Chem. 2009, 7 (9), 1753-1760). While this ‘OSMAC’ (‘One Strain ManyCompounds’) strategy is extremely useful in exploiting the fullbiosynthetic potential of a micro-organism of interest, it is ratherintensive in time and consumables. (Bode, 2002).

Rather than systematically changing culture conditions, the biosyntheticpotential of a micro-organism of interest can also be explored throughwhole genome sequencing. Many secondary metabolites are products ofknown biosynthetic pathways. The ability to ascribe a product to thegenes that code for it allows for the unique ability to analyze a wholegenome and predict the metabolites that can be produced. Cultureconditions curated to that biosynthetic pathway can then be employed toisolate specific compounds of interest. (Bromann, K.; Toivari, M.;Viljanen, K.; Vuoristo, A.; Ruohonen, L.; Nakari-Setälä, T.Identification and Characterization of a Novel Diterpene Gene Cluster inAspergillus nidulans. PLoS One 2012, 7 (4); Bergmann, S.; Schümann, J.;Scherlach, K.; Lange, C.; Brakhage, A. A.; Hertweck, C. Genomics—DrivenDiscovery of PKS-NRPS Hybrid Metabolites from Aspergillus nidulans. Nat.Chem. Biol. 2007, 3 (4), 213-217; Scherlach, K.; Hertweck, C. Discoveryof Aspoquinolones A-D, Prenylated Quinoline-2-One Alkaloids fromAspergillus nidulans, Motivated by Genome Mining. Org. Biomol. Chem.2006, 4, 3517-3520; Van Lanen, S. G.; Shen, B. Microbial Genomics forthe Improvement of Natural Product Discovery. Curr. Opin. Microbiol.2006, 9 (3), 252-260; Corre, C.; Challis, G. L. New Natural ProductBiosynthetic Chemistry Discovered by Genome Mining. Nat. Prod. Rep.2009, 26 (8), 977-986; Challis, G. L. Mining Microbial Genomes for NewNatural Products and Biosynthetic Pathways. Microbiology 2008, 154 (6),1555-1569).

Genome mining and the OSMAC approach are both useful techniques for thediscovery of the biosynthetic potential of a single organism. If,however, you have a microbial library that you would like to screen,these techniques may not be the most efficient. Epigeneticmodification—that is, the use of small molecule enzyme inhibitors topromote the expression and prevent the silencing or downregulation ofsecondary metabolite gene clusters can be used as a more ubiquitoustechnique to exploit the biosynthetic potential of a larger number ofmicroorganisms. (Williams, R. B.; Henrikson, J. C.; Hoover, A. R.; Lee,A. E.; Cichewicz, R. H. Epigenetic Remodeling of the Fungal SecondaryMetabolome. Org Biomol Chem 2008, 6 (11), 1895-1897; Cichewicz, R. H.Epigenome Manipulation as a Pathway to New Natural Product Scaffolds andTheir Congeners Robert. Nat. Prod. Rep. 2010, 27 (1), 11-22; Henrikson,J. C.; Hoover, A. R.; Joyner, P. M.; Cichewicz, R. H. A ChemicalEpigenetics Approach for Engineering the in Situ Biosynthesis of aCryptic Natural Product from Aspergillus niger. Org. Biomol. Chem. 2009,7 (3), 435-438; Wang, X.; Sena Filho, J. G.; Hoover, A. R.; King, J. B.;Ellis, T. K.; Powell, D. R.; Cichewicz, R. H. Chemical EpigeneticsAlters the Secondary Metabolite Composition of Guttate Excreted by anAtlantic-Forest-Soil-Derived Penicillium citreonigrum. J. Nat. Prod.2010, 73 (5), 942-948). Histone deacetylase (HDAC) and DNAmethyltransferase (DNMT) inhibitors can be used as culture additives toepigenetically ‘turn on’ secondary metabolite gene clusters in a libraryof filamentous fungi for the maximum surveying of bioactive naturalproduct potential therein. (Beau, J.; Mahid, N.; Burda, W. N.;Harrington, L.; Shaw, L. N.; Mutka, T.; Kyle, D. E.; Barisic, B.; VanOlphen, A.; Baker, B. J. Epigenetic Tailoring for the Production ofAnti-Infective Cytosporones from the Marine Fungus Leucostoma persoonii.Mar. Drugs 2012, 10 (4), 762-774).

There are many different techniques available for natural products drugdiscovery efforts. While exploring the biosynthetic potential of asingle organism can be very lucrative, screening efforts are needed inorder to identify those “lead” organisms. With a robustly designedscreening program, natural product extracts from a multitude of sourcescan be screened side by side in high-throughput capable bioassaysagainst a wide variety of disease targets. The resulting diversity ofbioactivity information combined with metabolite profiling can affordintense prioritization of extracts at a very early stage, streamliningfurther chemical investigation to a highly time and cost effective levelof efficiency.

Drug Discovery Targets

Natural products isolation efforts largely follow the same genericscheme (FIG. 1). Efforts aimed at drug discovery can take place at anyof the stages, from extraction to pure compound isolation. There arepros and cons to each approach, though it is generally accepted that theearlier the efforts can be prioritized, the better.

Crude extracts can contain thousands of compounds, however, it ispossible to get useful information from that complex mixture in ahigh-throughput way. Metabolite profiling of crude extracts can be usedfor initial dereplication and more advanced matabolomic analysis canreveal chemical outliers that may be of interest. (Sica, V. P.; Raja, H.A.; El-Elimat, T.; Kertesz, V.; Van Berkel, G. J.; Pearce, C. J.;Oberlies, N. H. Dereplicating and Spatial Mapping of SecondaryMetabolites from Fungal Cultures in Situ. J. Nat. Prod. 2015, 78 (8),1926-1936; Kellogg, J. J.; Todd, D. A.; Egan, J. M.; Raja, H. A.;Oberlies, N. H.; Kvalheim, O. M.; Cech, N. B. Biochemometrics forNatural Products Research: Comparison of Data Analysis Approaches andApplication to Identification of Bioactive Compounds. J. Nat Prod. 2016,79 (2), 376-386). High-throughput bioassays that are tolerant of complexmixtures can be used to discover and prioritize activity early in theinvestigation process. More sensitive and selective bioassays that arenot tolerant of complex mixtures would require more purified fractionsor pure compounds. It is important, therefore, when embarking on anatural products screening program, to coordinate bioassay capabilitiesto isolation protocols, in addition to other target selection criteria.The targets described below are of great contemporary relevance to humanhealth concerns and each feature robust bioassay methodologies thatassist in early crude extract level prioritization.

The ESKAPE Pathogens

With growing antibiotic resistance, and a decrease in antibiotic drugdiscovery, the Infectious Disease Society of America issued a ‘call toarms’ in 2009 to the drug discovery community to combat what they calledthe ESKAPE pathogens: the gram positive Enterococcus faecium andStaphylococcus aureus, and gram negative Klebsiella pneumoniae,Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobactercloacae. (Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.;Gilbert, D.; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J. BadBugs, No Drugs: No ESKAPE! An Update from the Infectious DiseasesSociety of America. Clin. Infect. Dis. 2009, 48 (1), 1-12). Theseclinically relevant, highly drug resistant pathogens represent acontinuously growing threat to human health and an important target fordrug discovery efforts. (Pogue, J. M.; Kaye, K. S.; Cohen, D. A.;Marchaim, D. Appropriate Antimicrobial Therapy in the Era ofMultidrug-Resistant Human Pathogens. Clin. Microbiol. Infect. 2015, 21(4), 302-312; Fleeman, R.; Lavoi, T. M.; Santos, R. G.; Morales, A.;Nefzi, A.; Welmaker, G. S.; Medina-Franco, J. L.; Giulianotti, M. A.;Houghten, R. A.; Shaw, L. N. Combinatorial Libraries as a Tool for theDiscovery of Novel, Broad-Spectrum Antibacterial Agents Targeting theESKAPE Pathogens. J. Med. Chem. 2015, 58 (8), 3340-3355).

Leishmania donovani

Cutaneous Leishmaniasis accounts for one million cases annually with 310million people being at risk for contraction. Visceral Leishmaniasisaccounts for 300,000 cases annually which result in 20,000 deathsannually. A neglected tropical disease (NTD), Leishmaniasis is aparasitic infection caused by an intramacrophage protozoa that istransmitted to humans by the bite of infected sandflies. The visceralform of this disease, most commonly caused by Leishmania donovani, istypically fatal when left untreated. Upon entering the host, theparasite—in a non-flagellated amastigote life stage—invades macrophagecells to travel through the body and reproduce. (Pulvertaft, R.; Hoyle,G. Stages in the Life-Cycyle of Leishmania donovani. Trans R Soc TropMed Hyg 1960, 54, 191-196). Recent advances in infected macrophagein-vitro culture techniques allow for more clinically relevant assays tobe performed in a high throughput screening (HTS) context. (Annang, F.;Perez-Moreno, G.; Garcia-Hernandez, R.; Cordon-Obras, C.; Martin, J.;Tormo, J. R.; Rodriguez, L.; de Pedro, N.; Gomez-Perez, V.; Valente, M.;Reyes, F.; Genilloud, O.; Vicente, F.; Castanys, S.; Ruiz-Perez, L. M.;Navarro, M.; Gamarro, F.; Gonzalez-Pacanowska, D. High-ThroughputScreening Platform for Natural Product-Based Drug Discovery against 3Neglected Tropical Diseases: Human African Trypanosomiasis,Leishmaniasis, and Chagas Disease. J. Biomol. Screen. 2015, 20 (1),82-91; De Rycker, M.; Hallyburton, I.; Thomas, J.; Campbell, L.; Wyllie,S.; Joshi, D.; Cameron, S.; Gilbert, I. H.; Wyatt, P. G.; Frearson, J.A.; Fairlamb, A. H.; Gray, D. W. Comparison of a High-ThroughputHigh-Content Intracellular Leishmania donovani Assay with an AxenicAmastigote Assay. Antimicrob. Agents Chemother. 2013, 57 (7), 2913-2922;Siqueira-Neto, J. L.; Moon, S.; Jang, J.; Yang, G.; Lee, C.; Moon, H.K.; Chatelain, E.; Genovesio, A.; Cechetto, J.; Freitas-Junior, L. H. AnImage-Based High-Content Screening Assay for Compounds TargetingIntracellular Leishmania donovani Amastigotes in Human Macrophages. PLoSNegl. Trop. Dis. 2012, 6 (6)). These advancements will hopefully aid inthe discovery of new treatments for this disease in the face ofincreasing resistance to existing treatments. (Balasegaram, M.;Ritmeijer, K.; Lima, M. A.; Burza, S.; Ortiz Genovese, G.; Milani, B.;Gaspani, S.; Potet, J.; Chappuis, F. Liposomal Amphotericin B as aTreatment for Human Leishmaniasis. Expert Opin. Emerg. Drugs 2012, 17(4), 493-510).

Mycobacterium tuberculosis

Tuberculosis (TB) remains a global health crisis, despite the advancesof the whole genome sequencing project that revealed the genome ofMycobacterium tuberculosis. This disease, whose latent form is estimatedto infect one third of the world's population, poses many drugdevelopment hurdles. Multi-drug resistant (MDR-TB) and extensively drugresistant (XDRTB) strains have emerged despite the current course oftreatment typically involving combinatorial therapies aimed directly atpreventing resistance. Drug discovery efforts, therefore, must addressnew mechanisms of action or M. tuberculosis targets. Additionally, TBdrugs have the burden of needing to be compatible in combinatorialtreatments for the immunocompromised, particularly those with HIV/AIDS,among whom incidence of this disease are highest. (Lechartier, B.;Rybniker, J.; Zumla, A.; Cole, S. T. Tuberculosis Drug Discovery in thePost-Post-Genomic Era. EMBO Mol. Med. 2014, 6 (2), 1-11). Naturalproducts based drug discovery against this target have revealedpromising results, with many existing treatments coming from naturalproducts. With such a demanding target comes the need to screen a broadswath of chemical space, confirming natural products drug discoveryefforts as a promising way forward in the search for treatments of thisdisease. (Mdluli, K.; Kaneko, T.; Upton, A. The Tuberculosis DrugDiscovery and Development Pipeline and Emerging Drug Targets. ColdSpring Harb Perspect Med 2015, 5).

Clostridium difficile

The leading cause of healthcare related infection, Clostridium difficileis an easily spread, diarrhea causing bacteria that is considered athreat to human health worldwide. The use of antibiotics which upset thehuman gut microbiome is the primary cause of C. difficile infection(CDI), but any immunocompromised individuals are at risk. Withincreasing incidences of resistance, recurrence, and mortality, the needfor discovery of new treatments against this bacteria is imperative.Most challengingly, new drugs to fight CDI must act without impact onthe normal human gut fauna. Many novel treatment avenues have beensuggested, among which, the discovery and use of bacterial naturalproducts remain of high interest. (Zucca, M.; Scutera, S.; Savoia, D.Novel Avenues for Clostridium difficile Infection Drug Discovery. ExpertOpin. Drug Discov. 2013, 8 (4), 459-477; Suwantarat, N.; Bobak, D. A.Current Status of Nonantibiotic and Adjunct Therapies for Clostridiumdifficile Infection. Curr. Infect. Dis. Rep. 2011, 13 (1), 21-27;Spigaglia, P. Recent Advances in the Understanding of AntibioticResistance in Clostridium difficile Infection. Ther. Adv. Infect. Dis.2016, 3 (1), 23-42).

Naegleria fowleri

Naegleria fowleri is a free living, warm-water loving amoeba that causesthe nearly always fatal primary amoebic meningoencephalitis (PAM).Diagnosis of PAM is extremely difficult, and current treatment optionsare time sensitive and limited to existing drug combinations (e.g.Amphotericin B, fluconazole, and miltefosine). (Capewell, L. G.; Harris,A. M.; Yoder, J. S.; Cope, J. R.; Eddy, B. A.; Roy, S. L.; Visvesvara,G. S.; Fox, L. M.; Beach, M. J. Diagnosis, Clinical Course, andTreatment of Primary Amoebic Meningoencephalitis in the United States,1937-2013; YODER, J. S.; EDDY, B. A.; VISVESVARA, G. S.; CAPEWELL, L.;BEACH, M. J. The Epidemiology of Primary Amoebic Meningoencephalitis inthe USA, 1962-2008. Epidemiol. Infect. 2010, 138 (7), 968-975). Asclinicians start to understand and diagnose PAM better, and the risk ofthis disease continues to rise with increasing global temperatures, newdrugs that specifically target this amoeba are urgently needed.

Cancer Targets

With structures as diverse as their targets, natural products have longplayed a role in the treatment of various cancers. (Newman, D. J.;Cragg, G. M. Natural Products as Sources of New Drugs from 1981 to 2014.J. Nat. Prod. 2016, 79 (3), 629-661; Wani, M. C.; Taylor, H. L.; Wall,M. E.; Coggon, P.; McPhail, A. T. Plant Antitumor Agents. VI. Isolationand Structure of Taxol, a Novel Antileukemic and Antitumor Agent fromTaxus brevifolia. J. Am. Chem. Soc. 1971, 93 (9), 2325-2327; Fenical,W.; Jensen, P.; Kauffman, C.; Mayhead, S.; Faulkner, D.; Sincich, C.;Rao, M.; Kantorowski, E.; West, L.; Strangman, W.; Shimizu, Y.; Li, B.;Thammana, S.; Drainville, K.; Davies-Coleman, M.; Kramer, R.; Fairchild,C.; Rose, W.; Wild, R.; Vite, G.; Peterson, R. New Anticancer Drugs fromCultured and Collected Marine Organisms. Pharm. Biol. 2003, 41 (sup1),6-14). As understanding of the complex physiology of human cells (bothhealthy and cancerous) continues to grow, assays directed at testingcompounds against highly specific cellular targets continue to emerge.Rather than whole cancer-cell assays, these target specific assays canhelp to exclude compounds that are broadly cytotoxic to all cells infavor of compounds that are active within the specific mechanism ofaction that is desired. Autopalmitoylation dysregulation is implicatedin many disease states. In a newly developed assay, palmitoylation ofproteins can be monitored for modulation by compounds of interest in aHTS manner. This allows compounds to be rapidly screened not for theireffect on the whole cell, but rather just on this particular pathway ofinterest. (Hamel, L. D.; Deschenes, R. J.; Mitchell, D. A. AFluorescence-Based Assay to Monitor Autopalmitoylation of zDHHC ProteinsApplicable to High-Throughput Screening Q. Anal. Biochem. 2014, 460,1-8).

SUMMARY OF INVENTION

Fungi are known to produce a wide range of secondary metabolites ofinterest in drug discovery efforts. In a search for new, bioactivenatural products via a fungal metabolite screening program, five newcompounds (1-5) were isolated. Bioactivities of the new compoundsagainst various drug targets make these compounds and their derivativesof interest for further drug discovery efforts.

The present invention relates to compounds according to the structure:

where R1 can be

where R2 is a hydroxyl group.

where R3 can be, or

where R4 can be

where R5 is

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1 is an image depicting a flowchart for natural product isolation.

FIG. 2 is an image depicting an extraction scheme for Phomopsis sp.Control.

FIG. 3 is an image depicting chemical structures of new compounds 1-5and known compound 6.

FIG. 4 is an image depicting NP HPLC chromatogram of fraction D_C (FIG.2) from the control growth treatment of Phomopsis sp. Peaks areannotated with their isolated compounds (FIG. 3). Each compound requiredsome further purification on reverse phase (RP) HPLC for structureelucidation and bioassay purposes, but represented the major componentof each of the peaks seen. Black is the UV chromatogram at 320 nm, andblue is the ELSD trace.

FIG. 5 is an image depicting important COSY, HMBC, and NOE correlationsin phomopsichromin A (1).

FIG. 6 is an image depicting important COSY, HMBC, and NOE correlationsin phomopsichromin B (2).

FIG. 7 is an image depicting methylation reaction of 2 to yield 7 withdiazomethane.

FIG. 8 is an image depicting important COSY, HMBC, and NOE correlationsin phomopsichromin C (3).

FIG. 9 is an image depicting the proposed biosynthetic pathway towardscyclohexane substructure of 1-6 from Tanabe and Suzuki, 1974.

FIG. 10 is an image depicting proposed stereocenters in the cyclohexanering of 3 sent for ECD calculations.

FIG. 11 is an image depicting important COSY, HMBC, and NOE correlationsin phomopsichromin D (4).

FIG. 12 is an image depicting important COSY and HMBC correlations inphomopsichromin E (5).

FIG. 13A-B is a series of images depicting a) Representative pie chartfrom bioassay data, in this case, ESKAPE activity, that shows theactivity boosting effects of the epigenetic modification; b)Representative pie chart from bioassay data, in this case, ESKAPEactivity, that demonstrates distribution of activity across the threetreatment conditions. This is the overall trend amongst all testingcompleted to date.

FIG. 14 is an image depicting selectivity of active extracts.

FIG. 15 is a series of images of previously reported citreohybridones,citreohybriddiones A-C, and the new citreohybriddione D (1).

FIG. 16 is an image depicting the NP MPLC chromatogram of KML12-14MG-B2aHDACi.

FIG. 17 is an image depicting the NP HPLC chromatogram of KML12-14MG-B2aHDACi-F.

FIG. 18 is an image depicting important COSY, HMBC, and NOESYcorrelations in Citreohybriddione D (1).

FIG. 19 is an image depicting a 3D depiction of 1 for stereochemicalreview.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following detailed description of the preferred embodiments,reference is made to the accompanying drawings, which form a parthereof, and within which are shown by way of illustration specificembodiments by which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the invention.

Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are described herein. All publicationsmentioned herein are incorporated herein by reference in their entiretyto disclose and describe the methods and/or materials in connection withwhich the publications are cited. It is understood that the presentdisclosure supercedes any disclosure of an incorporated publication tothe extent there is a contradiction.

All numerical designations, such as pH, temperature, time,concentration, and molecular weight, including ranges, areapproximations which are varied up or down by increments of 1.0 or 0.1,as appropriate. It is to be understood, even if it is not alwaysexplicitly stated that all numerical designations are preceded by theterm “about”. It is also to be understood, even if it is not alwaysexplicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art and can besubstituted for the reagents explicitly stated herein.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit, unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassed inthe invention. The upper and lower limits of these smaller ranges mayindependently be excluded or included within the range. Each range whereeither, neither, or both limits are included in the smaller ranges arealso encompassed by the invention, subject to any specifically excludedlimit in the stated range. Where the stated range includes one or bothof the limits, ranges excluding either or both of those excluded limitsare also included in the invention.

The term “about” as used herein refers to being within an acceptableerror range for the particular value as determined by one of ordinaryskill in the art, which will depend in part on how the value is measuredor determined, i.e. the limitations of the measurement system, i.e. thedegree of precision required for a particular purpose. In general, theterm “about” refers to being approximately or nearly and in the contextof a numerical value or range set forth means ±15% of the numericalvalue.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise.

Compounds described herein can contain one or more double bonds and,thus, potentially give rise to cis/trans (E/Z) isomers, as well as otherconformational isomers. Unless stated to the contrary, the inventionincludes all such possible isomers, as well as mixtures of isomers.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g. each enantiomer and diastereomer, and a mixture ofisomers, such as racemic or scalemic mixture. Compounds described hereincan contain one or more asymmetric centers and, thus potentially giverise to diastereomers and optical isomers. Unless stated to thecontrary, the present invention includes all such possible diastereomersas well as their racemic mixtures, their substantially pure resolvedenantiomers, all possible geometric isomers, and pharmaceuticallyacceptable salts thereof. Mixtures of stereoisomers, as well as isolatedspecific stereoisomers, are also included.

“Terpene” as used herein refers to organic hydrocarbons built up fromisoprene, a hydrocarbon consisting of five carbon atoms attached toeight hydrogen atoms (C₅H₈). Terpenes have a general formula of (C₅H₈)n.“Terpenoids” refers to oxygenated derivatives of these hydrocarbons.

New Antimicrobials

Chemistry for terpenoid metabolites can be found in Ellestad, G. A. etal, 1969, herein incorporated by reference into this disclosure in itsentirety (Ellestad, G. A., et al., Some new terpenoid metabolites froman unidentified fusarium species, Tetrahedron, 1969, 25(6): 1323-1334).Sesquiterpene hydroquinones and isolation thereof can be found in Shenet al. 2001, herein incorporated by reference by its entirety. (Shen, Y.C., et al., New sesquiterpene hydroquinones from a Taiwanese marinesponge Hippospongia metachromia, 2001, J. Nat. Prod., 64:801-803).

BB11-2 Isolation

Small colonies of the fresh water bryozoan Pectinatella magnifica werenoticed in a retention pond located in north Tampa, Fla. These colonialorganisms were collected for chemical analysis. While collecting, one ofthe colonies was found to be growing around the end of a tree branchthat was partially submerged in the water. A small piece of this branchwas collected along with the bryozoan and returned to the lab forprocessing. Once in the lab, the branch was processed for microbialisolation as discussed herein.

From two SDA plates, 7 fungal isolates were obtained based onmorphological features. These organisms were given a name of “BB11” andarchived. Following chemical investigation of isolate BB11-2, fungalidentification was obtained via Sanger sequencing of the 18S ribosomalspacer region. Forward primers for the 18S region are as follows: 5′NNNNNNNNNNNNNTTGGTTTCTAGGACCGCCGTAATGATTAATAGGGACAGTCGGGGGCATCAGTATTCAATCGTCAGAG 3′ (SEQ ID NO:1); 5′GTGAAATTCTTGGATCGATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAGGAACGAAAGT 3′ (SEQ ID NO:2); 5′TAGGGGATCGAAAACGATCAGATACCGTTGTAGTCTTAATCATAAACTATGCCCACTAGGGATCNGGCGGTGTTATTTCT 3′ (SEQ ID NO:3). Reverse primers are asfollows: 5′ NNNNNNNNNNNNNNNCNGNTCNCCCCTTGTGGTGCCCTTCCGTCAATTTCTTTAAGTTTCAGCCTTGCGACCATACTCCC 3′ (SEQ ID NO:4); 5′CCCAGAACCCAAAAACTTTACTTTCGTGTAAGGTGCCGAGCGGGTCAAGAAATAACACCGCCCGATCCCTAGTCGGCATA 3′ (SEQ ID NO:5); 5′GTTTATGGTTAAGACTACAACGGTATCTGATCGTTTTCGATNCCCTAACTTTCGTTCCTGATTNANGANAACATCCTTGG 3′ (SEQ ID NO:6); 5′GAAATGCTTTCCNANTAATNNGNCTTCNATCAAATCCTCA 3′ (SEQ ID NO:7). An NCBInucleotide BLAST search returned identification as Diaporthe orPhomopsis sp.; genera that have been determined to be the same butrepresent different life cycles of these common, chemically richendophytic fungi. (Gomes, R. R.; Glienke, C.; Videira, S. I. R.;Lombard, L.; Groenewald, J. Z.; Crous, P. W. Diaporthe: A Genus ofEndophytic, Saprobic and Plant Pathogenic Fungi. Persoonia Mol.Phylogeny Evol. Fungi 2013, 31, 1-41; Dai, J.; Krohn, K.; Flörke, U.;Gehle, D.; Aust, H. J.; Draeger, S.; Schulz, B.; Rheinheimer, J. NovelHighly Substituted Biraryl Ethers, Phomosines D-G, Isolated from theEndophytic Fungus Phomopsis Sp. from Adenocarpus Foliolosus. European J.Org. Chem. 2005, 4 (23), 5100-5105; Kobayashi, H.; Meguro, S.;Yoshimoto, T.; Namikoshi, M. Absolute Structure, Biosynthesis, andAnti-Microtubule Activity of Phomopsidin, Isolated from a Marine-DerivedFungus Phomopsis Sp. Tetrahedron 2003, 59 (4), 455-459; Isaka, M.;Jaturapat, A.; Rukseree, K.; Danwisetkanjana, K.; Tanticharoen, M.;Thebtaranonth, Y. Phomoxanthones A and B, Novel Xanthone Dimers from theEndophytic Fungus Phomopsis Species. J. Nat. Prod. 2001, 64 (8),1015-1018; Tang, J. W.; Wang, W. G.; Li, A.; Yan, B. C.; Chen, R.; Li,X. N.; Du, X.; Sun, H. D.; Pu, J. X. Polyketides from the EndophyticFungus Phomopsis Sp. sh917 by Using the One Strain/many CompoundsStrategy. Tetrahedron 2016, 10, 2-9; Lin, X.; Huang, Y.; Fang, M.; Wang,J.; Zheng, Z.; Su, W. Cytotoxic and Antimicrobial Metabolites fromMarine Lignicolous Fungi, Diaporthe Sp. FEMS Microbiol. Lett. 2005, 251(1), 53-58; Dettrakul, S.; Kittakoop, P.; Isaka, M.; Nopichai, S.;Suyarnsestakorn, C.; Tanticharoen, M.; Thebtaranonth, Y.Antimycobacterial Pimarane Diterpenes from the Fungus Diaporthe Sp.Bioorganic Med. Chem. Lett. 2003, 13 (7), 1253-1255). The isolate isreferred to herein as Phomopsis sp.

Initial Bioactivities and Scale-Up

The BB11 fungal isolates were screened in a number of assays and assaydevelopment protocols and their extracts were known within the lab to behighly active with low cytotoxicity. Isolate BB11-2 was chosen for itsreproducible bioactivity and tolerance towards multiple media types. Asa part of a scale-up optimization study, BB11-2 was scaled up on ricemedia in three growth conditions: control (700 g of rice media+100 mLSDB), HDACi (700 g of rice media+a 435 μM solution of sodium butyrate in100 mL SDB), and DNMTi (700 g of rice media+a 435 μM solution of5-azacytidine in 100 mL SDB). After 21 days of growth, each extract wasextracted in 1:3 MeOH/EtOAc solution overnight, followed by 2 subsequent24 hour extractions in 100% EtOAc. The extracts for each of the threeculture conditions were dried down and subjected to a H2O:EtOAcpartition. The lipophilic partitions were each separated on NP MPLC(FIG. 2). The fractions of the control extract were furtherinvestigated.

Compound Isolation and Structure Elucidation

The extract of the control culture condition of Phomopsis sp. wasinvestigated by NMR and bioactivity guided fractionation (FIG. 2) andyielded 5 new meroterpenes: phomopsichromins A-E (1-5), and the knowncompound LL-Z1272ε (6) (FIG. 3). (Ellestad, G. A.; Evans, R. H.;Kunstmann, M. P. Some New Terpenoid Metabolites from an UnidentifiedFusarium Species. Tetrahedron 1969, 25 (6), 1323-1334).

In the first MPLC separation of the lipophilic partition of the Controlextract, the majority of the mass eluted in one large peak inapproximately 1:1 n-hexanes:ethyl acetate. This fraction (Fraction D)was further purified via NP MPLC on an extended non-polar gradient(n-hexanes to ethyl acetate). Again, the majority of the mass eluted ina single peak in approximately 1:1 nhexanes:ethyl acetate (Fraction C).Further purification was accomplished on NP HPLC using a cyano column(CN capping of the silica particles) and UV detection. Compounds 1-6(FIG. 3) were isolated as illustrated in FIG. 2.

Compounds 1-6 all share a sesquiterpene backbone functionalizeddifferentially at C-9. 1-3 share a chromene substructure while compounds4-6 feature a ring-opened subunit. The structures of PhomopsichrominsA-E (1-5) were elucidated as described below.

Phomopsichromin A (1)

Phomopsichromin A (1), [α]²⁰D +1.2, was isolated as a white powder. The1H NMR spectrum of 1 (Table 1) notably displayed 3 olefinic protons,peaks for 5 methyl substituents, and a phenol. The 13C NMR spectrumindicated the presence of a carboxylic acid (δC 175.18) and ketone (δC213.87). Remaining 13C signals were split between the aromatic andalkene regions and implied a high level of substitution on the aromaticring (as evidenced by the small number of olefinic protons in the 1H NMRspectrum). The chromene substructure was tentatively assigned based on2D NMR experiments and 13C ppm shifts of carbons 9 and 21. The remainingterpene scaffold was completed based on HMBC and COSY NMR data (FIG. 5).HRESIMS of 1 (m/z 387.2137 [M+H]+) resembled compounds in the literaturebut did not match any exactly, confirming that 1 was a new compound.(Ellestad, G. A.; Evans, R. H.; Kunstmann, M. P. Some New TerpenoidMetabolites from an Unidentified Fusarium Species. Tetrahedron 1969, 25(6), 1323-1334; Singh, S. B.; Zink, D. L.; Bills, G. F.; Jenkins, R. G.;Silverman, K. C.; Lingham, R. B. Cylindrol A: A Novel Inhibitor of RasFarnesyl-Protein Transferase from Cylindrocarpon Lucidum. TetrahedronLett. 1995, 36 (28), 4935-4938; Singh, S. B.; Ball, R. G.; Bills, G. F.;Cascales, C.; Gibbs, J. B.; Goetz, M. A.; Hoogsteen, K.; Jenkins, R. G.;Liesch, J. M.; Lingham, R. B.; Silverman, K. C.; Zink, D. L. Chemistryand Biology of Cylindrols: Novel Inhibitors of Ras Farnesyl-ProteinTransferase from Cylindrocarpon Lucidum. J. Org. Chem. 1996, 61 (22),7727-7737; Ishibashi, M.; Ohizumi, Y.; Cheng, J.; Nakamura, H.; Sasaki,T.; Kobayashi, J. Metachromins A and B, Novel AntineoplasticSesquiterpenoids from the Okinawan Sponge Hippospongia Cf. Metachromia.J. Org. Chem. 1988, 53 (12), 2855-2858; Takahashi, Y.; Yamada, M.;Kubota, T.; Fromont, J.; Kobayashi, J. Metachromins R--T, NewSesquiterpenoids from Marine Sponge Spongia Sp. Chem. Pharm. Bull.(Tokyo). 2007, 55 (12), 1731-1733).

TABLE 8 1D and 2D data for phomopsichromin A (1) in CDCl₃. Pos δ_(C)^(b) δ_(H) (m_(,) J(HZ))^(a) HMBC^(c) 1 50.4 2.41 (q, 6.6 × 2, 1 H) 2,5, 6, 7, 12, 14 2 213.8 3 41.5 2.33 (m, 1 H) 2, 4, 5 2.36 (m, 1 H) 430.8 1.64 (m, 1 H) 3, 5, 6, 13 1.85 (m, 1 H) 2, 3, 5, 6 5 36.1 1.97 (m,1 H) 1, 3, 4, 6, 13 6 43.2 7 30.6 1.41 (m, 1 H) 1, 5, 6, 8, 14 1.48 (m,1 H) 1, 5, 6, 8, 14 8 34.3 1.52 (m, 1 H) 7, 9, 10 1.77 (m, 1 H) 7, 15,9, 10 9 79.89 10 125.8 5.46 (d, 10.2 1 H) 8, 9, 15, 16, 21 11 116.9 6.76(d, 10.2 1 H) 9, 15, 16, 17, 21 12 7.5 0.91 (d, 6.8, 3 H) 1, 2, 6 1314.9 0.89 (d, 6.8, 3 H) 4, 5, 6 14 15.4 0.59 (s, 3 H) 1, 5, 6, 7 15 271.44 (s, 3 H) 8, 9, 10, 11 16 106.8 17 160.6 —OH 11.74 (s, 1 H) 16, 17,18, 21 18 103.6 19 144.4 20 111.9 6.24 (s, 1 H) 10, 11, 16, 18, 21, 22,23 21 158.6 22 24 2.55 (s, 3 H) 17, 18, 19, 20, 21, 23 23 175.1 ^(a1)HNMR recorded at 500 MHz, reported in ppm (multiplicity, J-coupling inHz, integration); ^(b13)C NMR recorded at 200 MHz; ^(c)recorded from agHMBCAD experiment at 500 MHz and reported as positions of carbons.

Compound data for Phomopsichromin A (1) is as follows: C₂₃H₃₀O₅; HRESIMSm/z 369.2034 [M+H−H₂O]⁺ (C₂₃H₂₉O₄ calculated, 369.2066), m/z 387.2137[M+H]⁺ (C₂₃H₃₁O₅ calculated, 387.2171), m/z 409.1954 [M+Na]⁺ (C₂₃H₃₀O₅Nacalculated, 409.1991); UV (MeOH) λmax (log ε) 250 (4.97) nm; [α]²⁰_(D)+1.2 (c 0.1, MeOH); IR (thin film) 3450, 2980, 2360, 1700, 1600,1575, 1450, 1400, 1300, 1090 cm⁻¹; H NMR Data (500 MHz, CDCl₃) δ ppm0.59 (s, 3H), 0.89 (d, J=6.8 Hz, 3H), 0.91 (d, J=6.8 Hz, 3H), 1.41 (m,1H), 1.44 (s, 3H), 1.48 (m, 1H), 1.52 (m, 1H), 1.64 (m, 1H), 1.77 (m,1H), 1.85 (m, 1H), 1.97 (m, 1H), 2.33 (m, 1H), 2.36 (m, 1H), 2.41 (q,J=6.6 Hz, 1H), 2.55 (s, 3H), 5.46 (d, J=10.2 Hz, 1H), 6.24 (s, 1H), 6.76(d, J=10.2 Hz, 1H), 11.74 (s, 1H); ¹³C NMR (125 MHz, CDCl₃) δ ppm 7.5(CH₃, C-12), 14.9 (CH₃, C-13), 15.4 (CH₃, C-14), 30.6 (CH₂, C-7), 30.8(CH₂, C-4), 34.3 (CH₂, C-8), 36.1 (CH, C-5), 41.5 (CH₂, C-3), 43.2(C-6), 50.4 (CH, C-1), 79.8 (C-9), 103.6 (C-18), 106.8 (C-16), 111.9(CH, C-20), 116.9 (CH, C-11), 125.8 (CH, C-10), 144.4 (C-19), 158.6(C-21), 160.6 (C-17), 175.1 (C-23) 213.8 (C-2).

Using the NMR data from these related compounds, the chromenesubstructure was confirmed. Relative stereochemical assignments atmethyl-bearing carbons 1 (R), 5 (R), 6 (S), and 9 (S) were assigned via1 and 2D NOE experiments (FIG. 5) and comparison to the literature.(Ishibashi, M.; Ohizumi, Y.; Cheng, J.; Nakamura, H.; Sasaki, T.;Kobayashi, J. Metachromins A and B, Novel AntineoplasticSesquiterpenoids from the Okinawan Sponge Hippospongia Cf. Metachromia.J. Org. Chem. 1988, 53 (12), 2855-2858; Takahashi, Y.; Yamada, M.;Kubota, T.; Fromont, J.; Kobayashi, J. Metachromins R--T, NewSesquiterpenoids from Marine Sponge Spongia Sp. Chem. Pharm. Bull.(Tokyo). 2007, 55 (12), 1731-1733; Tanabe, M.; Suzuki, K. Detection ofC—C Bond Fission during the Biosynthesis of the Fungal TriprenylphenolAscochlorin Using [1,2-13C]-Acetate. J.C.S. Chem. Comm. 1974, No. 1,445-446). ECD will be used to confirm absolute stereochemistry.

Phomopsichromin B (2)

Phomopsichromin B (2) was isolated as a white powder, C23H32O5, HRESIMSm/z 389.2322 [M+H]+ (calculated, 389.2283), [α]²⁰D −1.5. Comparison tothe HRMS of 1 indicated a difference of 2 protons. The appearance of abroad multiplet at δH 3.85 and the loss of the ketone signal at δC213.87 inferred a reduction at C-2. The 2D NMR spectra further supportedthis assignment (FIG. 6). The stereochemistry at position 2 wasdetermined to be S based on the multiplicity observed in the proton NMRspectrum. The observed J-values of the quartet at δH 3.85 most closelymatched those expected from the axial orientation of the hydroxyl group.

TABLE 2 1D and 2D data for phomopsichromin B (2) in CDCl₃. Pos δ_(C)^(b) δ_(H) (m, J(Hz))^(a) HMBC^(c) 1 39.4 1.43 (m, 1H) 2, 6, 12, 14 273.1 3.85 (q, 2.8, 1H) 3, 4, 6, 12 3 33.9 1.55 (m, 1H) 1, 5 1.8 (m, 1H)1, 2, 4, 5 4 25.4 1.27 (m, 1H) 1.63 (m, 1H) 3, 5 5 36.5 1.46 (m, 1H) 6,7, 14 6 38.2 7 30.9 1.35 (m, 2H) 5, 6, 8, 14 8 34.4 1.45 (m, 1H) 6, 7, 91.63 (m, 1H) 6, 7. 9, 10, 15 9 80.1 10 126.2 5.46 (d, 10.2, 1H) 8, 9,15, 16 11 116.6 6.74 (d, 10.2, 1H) 9, 16, 17, 21 12 12.3 0.95 (d, 7.1,3H) 1, 2, 6 13 15.6 0.82 (d, 6.6, 3H) 4, 5, 6 14 17.3 0.86 (s, 3H) 7, 6,5 15 27.1 1.41 (s, 3H) 8, 9, 10 16 106.9 17 160.6 —OH 11.80 (s, 1H) 16,17, 18 18 103.4 19 144.1 20 111.9 6.22 (s, 1H) 16,18, 21, 22 21 158.7 2224.4 2.53 (s, 3H) 18, 19, 20 23 174.7 ^(a1)H NMR recorded at 600 MHz,reported in ppm (multiplicity, J-coupling in Hz, integration); ^(b13)CNMR recorded at 125 MHZ; ^(c)recorded from a gHMBCAD experiment at 500MHz and reported as positions of carbons.

This orientation could also be observed in other related compounds inthe literature. (Bieber, L. W.; Messana, I.; Lins, S. C.; Da SilvaFilho, A. A.; Chiappeta, A. A.; De Mello, J. F. MeroterpenioidNaphthoquinones from Cordia Corymbosa. Phytochemistry 1990, 29(6),1955-1959). The remaining chiral centers were determined to be the sameas in 1. Again, the stereochemistry is confirmed via ECD.

Curiously, in multiple 1D NMR analyses of 2 over time, notable shifts inppm were observed for some peaks (e.g. δH 6.22, 3.85, and 2.53; δC73.17, 103.46, 158.73, and 174.72). The assumption was made that therewere diastereoisomers present (further supported by some poorly resolvedcrystal data), however, various NP and RP HPLC attempts eluted a singlepeak in all conditions. The beginning, middle, and end of the peak werecollected as separate fractions (a, b, and c) in an attempt to separatethe diastereoisomers. Clear ppm differences were observed betweenfractions a and c, but they were chromatographically indistinguishable.The decision was made to methylate the carboxylic acid at C-23 infractions a and c to reduce the effects of hydrogen bonding inseparation attempts and aid in NMR spectral evaluation (FIG. 7).

The resulting methyl derivatives of fractions a and c (7) were confirmedby 1D NMR. All ppm differences between fractions a and c were lost,indicating that all previously noted shifts in ppm had been aconcentration-based artifact of hydrogen bonding of 2 to itself insolution. 2 was determined to be a single diastereoisomer and remainingchemical and biological analyses were completed.

Compound data for Phomopsichromin B (2) is as follows: C₂₃H₃₂O₅; HRMSm/z 371.2222 [M+H−H₂O]⁺ (C₂₃H₃₁O₄ calculated, 371.2222), m/z 389.2322[M+H]⁺ (C₂₃H₃₃O₅ calculated, 389.2328), m/z 411.2145 [M+Na]⁺ (C₂₃H₃₂O₅Nacalculated, 411.2147); UV (MeOH) λmax (log ε) 250 (4.08) nm; [α]²⁰D −1.5(c 0.1, MeOH); IR (thin film) 3450, 2930, 2380, 1675, 1625, 1600, 1575,1460, 1400, 1300, 1090 cm⁻¹; ¹H NMR Data (600 MHz, CDCl₃) δ ppm 0.82 (d,J=6.6 Hz, 3H), 0.86 (s, 3H), 0.95 (d, J=7.1 Hz, 3H), 1.27 (m, 1H), 1.35(m, 2H), 1.41 (s, 3H), 1.43 (m, 1H), 1.45 (m, 1H), 1.46 (m, 1H), 1.55(m, 1H), 1.63 (m, 2H), 1.8 (m, 1H), 2.53 (s, 3H), 3.85 (q, 2.8, 1H),5.46 (d, J=10.2 Hz, 1H), 6.22 (s, 1H), 6.74 (d, J=10.2 Hz, 1H), 11.80(s, 1H); ¹³C NMR (125 MHz, CDCl₃) δ ppm 12.3 (CH₃, C-12), 15.6 (CH₃,C-13), 17.3 (CH₃, C-14), 24.4 (CH₃, C-22), 25.4 (CH₂, C-4), 27.0 (CH₃,C-15), 30.9 (CH₂, C-7), 33.9 (CH₂, C-3), 34.4 (CH₂, C-8), 36.5 (CH,C-5), 38.2 (C-6), 39.4 (CH, C-1), 73.1 (CH, C-2), 80.1 (C-9), 103.4(C-18), 106.9 (C-16), 111.9 (CH, C-20), 116.6 (CH, C-11), 126.2 (CH,C-10), 144.1 (C-19), 158.7 (C-21), 160.6 (C-17), 174.7 (C-23).

Phomopsichromin C (3)

Phomopsichromin C (3) was isolated as a white powder, [α]20D +1.3. 1HNMR data resembled 1 and 2 in the chromene region, however, the methylsignals at position 12, 13, and 14 were notably shifted downfield andwere all overlapping (Table 3). New peaks at δC 170.87 and 21.4, with anew methyl signal in the 1H spectrum (δH 2.05) indicated added esterfunctionalization in the molecule. HRESIMS m/z 431.2434 [M+H]+(calculated, 431.2389) gave a molecular formula of C25H34O6, confirmingthe addition of a —CO(O)CH3 group. The available 2D NMR data confirmedthat the new functionalization was at C-2 (δC 75.16) (FIG. 8).

TABLE 3 1D and 2D data for phomopsichromin C (3) in CDCl₃. Pos δ_(C)^(b) δ_(H) (m, J(Hz))^(a) HMBC^(c) 1 38.5 1.55 (m, 1H) 6, 12, 14 2 75.14.97 (q, 2.0, 1H) 1 3 30.9 1.5 (m, 1H) 1, 2, 4, 5 1.81 (m 1H) 4 25.81.29 (m, 1H) 2, 3, 5, 6, 13 1.5 (m, 1H) 5 36.2 1.47 (m, 1H) 3, 4. 6, 146 38.3 7 30.7 1.4 (m, 2H) 1, 5, 8, 9, 14 8 34.4 1.4 (m, 1H) 7, 9, 10,11, 15 1.65 (m, 1H) 9 80.1 10 126.1 5.45 (d, 10.2, 1H) 8, 9, 15, 16, 2111 116.7 6.74 (d, 10.0, 1H) 9, 15-17, 21 12 12.0 0.83 (m, 3H) 2 13 15.60.83 (m, 3H) 4 14 16.7 0.83 (m, 3H) 1, 5, 7, 8 15 27.1 1.4 (s, 3H) 8-1116 106.8 17 160.6 —OH 11.82 (s, 1H) 16, 17, 18, 21 18 103.3 19 144.1 20111.9 6.22 (s, 1H) 11, 16-18, 21-23 21 158.7 22 24.4 2.53 (s, 3H) 16-20,23 23 174.4 24 170.8 25 21.4 2.05 (s, 3H) 2, 24 ^(a1)H NMR recorded at600 MHz, reported in ppm (multiplicity, J-coupling in Hz, integration);^(b13)C NMR recorded at 125 MHz; ^(c)recorded from a gHMBCAD experimentat 500 MHz and reported as positions of carbons.

Due to the overlapping signals of the methyl signals on the cyclohexanering of 3, elucidating the stereochemistry at C-1, 2, 5, and 6 based onNMR data posed a significant challenge. The methyl group at C-9 wasassumed, as in compounds 1 and 2, to be S based on chemical shift. Thestereochemistry at C-2 was again determined to be S based on the 1D 1Hmultiplicity. FIG. 9 illustrates the proposed biosynthetic pathway forthe cyclohexane substructure of 1-5 based on work done by Tanabe andSuzuki in 1974 on a related compound. 13 This pathway indicates that theketone at C-2 is a part of the precursor molecule, and therefore allreductions at C-2 occur post translationally. This suggests a conservedchirality at the centers at C-1, C-5, and C-6. Based on theseassumptions, a compound with proposed stereochemistry (FIG. 10) wassubmitted for ECD.

Compound data for Phomopsichromin C (3): C₂₅H₃₄O₆; HRESIMS m/z 413.2330[M+H−H₂O]⁺ (C₂₅H₃₃O₅ calculated, 413.2328), m/z 431.2434 [M+H]⁺(C₂₅H₃₅O₆ calculated, 431.2434), m/z 453.2256 [M+Na]⁺ (C₂₅H₃₄O₆Nacalculated, 453.2253); UV (MeOH) λmax (log ε) 255 (4.21) nm; [α]²⁰_(D)+1.3 (c 0.1, MeOH); IR (thin film) 2930, 1740, 1675, 1600, 1580,1400, 1300, 1350, 1090 cm⁻¹; ¹H NMR Data (600 MHz, CDCl₃) δ ppm 0.83(2d, J=6.6 Hz, 6H; s, 3H), 1.29 (m, 1H), 1.40-1.43 (m, 6H), 1.47 (m,1H), 1.5 (m, 2H), 1.55 (m, 1H), 1.65 (m, 1H), 1.81 (m, 1H), 2.05 (s,3H), 2.53 (s, 3H), 4.97 (q, 2.0, 1 H), 5.45 (d, J=10.2 Hz, 1H), 6.22 (s,1H), 6.74 (d, J=10.0 Hz, 1H), 11.82 (s, 1H); ¹³C NMR (125 MHz, CDCl₃) δppm 12.0 (CH₃, C-12), 15.6 (CH₃, C-13), 16.7 (CH₃, C-14), 21.4 (CH₃,C-25), 24.4 (CH₃, C-22), 25.8 (CH₂, C-4), 27.1 (CH₃, C-15), 30.7 (CH₂,C-7), 30.9 (CH₂, C-3), 34.4 (CH₂, C-8), 36.2 (CH, C-5), 38.3 (C, C-6),38.5 (CH, C-1), 75.1.

Phomopsichromin D (4)

Phomopsichromin D (4), C23H34O5, HRESIMS m/z 391.2473 [M+H]+(calculated, 391.2440), [α]²⁰D +0.2) was isolated as a white powder fromthe last and least lipophilic HPLC fraction of MPLC fraction D_C (FIG.4). 4 was notably distinct from 1-3 in that it was quite different inpolarity (not soluble in CDCl₃) and so all NMR data was obtained inDMSO-d6 (Table 4). The olefin region of the proton NMR spectrum of 4 wasthe most drastically different from compounds 1-3 and there was anadditional phenol signal at δH 10.02, indicating that 4 lacked thecompleted chromene substructure.

TABLE 4 1D and 2D data for phomopsichromin D (4) in DMSO-d₆. Pos δ_(C)^(b) δ_(H) (m₂ J(Hz))^(a) HMBC^(c) 1 39.0 1.35 (m, 1 H) 2, 5, 6, 12, 142 70.4 3.62 (q, 2.4, 1 H) 4, 6, 12 3 34.1 1.4 (m, 1 H) 4 1.6 (m, 1 H) 44 25.5 1.13 (m, 1 H) 1, 3, 5, 6, 14 1.53 (m, 1 H) 5 36.0 1.4 (m, 1 H) 46 38.0 7 36.0 1.17 (m, 1 H) 1, 5, 6, 8, 9, 14 1.23 (m, 1 H) 5, 6, 8, 9,14 8 32.4 1.69 (m, 1 H) 5, 15 1.75 (m, 1 H) 5, 10, 11, 15 9 134.7 10121.8 5.12 (br t, 6.9, 1 H) 8, 11, 15, 16 11 21.5 3.16 (br d, 7.2, 1 H)7, 8, 9, 10, 15, 16, 17, 21 12 12.7 0.84 (d, 7.2, 3 H) 1, 2, 6 13 15.70.74 (d, 6.6, 3 H) 4 14 17.3 0.75 (s, 3 H) 1, 6, 7 15 16.1 1.70 (s, 3 H)7, 8, 9, 10, 11, 16 16 112.0 17 159.6 —OH 12.61 (s, 1 H) 16, 17, 18, 2118 103.6 19 139.8 20 110.4 6.23 (s, 1 H) 11, 16, 17, 18, 21, 22, 23 21162.8 —OH 10.02 (s, 1 H) 16, 17, 20, 21 22 23.8 2.38 (s, 3 H) 18, 19,20, 21, 23 23 174.1 ^(a1)H NMR recorded at 500 MHZ, reported in ppm(multiplicity. J-coupling in Hz integration); ^(b13)C NMR reocrded at125 MHZ; ^(c)recorded from a gHMBCAD experiment at 500 MHz and reportedas positions of carbons.

2D NMR data (FIG. 11) further supported this, and the “open” carbonskeleton was assigned and confirmed by comparison to literature data onknown compound 6. Stereochemistry on the cyclohexane ring was set as incompounds 1-3. The double bond of the open chain was determined to be Ebased on the comparison of the J-value of H-10 to the literature.

Compound data for Phomopsichromin D (4) is as follows: C₂₃H₃₄O₅; HRESIMSm/z 373.2373 [M+H−H₂O]⁺ (C₂₃H₃₃O₄ calculated, 373.2379), m/z 391.2473[M+H]⁺ (C₂₃H₃₅O₅ calculated, 391.2484), m/z 413.2298 [M+Na]⁺ (C₂₃H₃₄O₅Nacalculated, 413.2304); UV (MeOH) λmax (log ε) 225 (5.89) nm; [α]²⁰_(D)+0.2 (c 0.1, MeOH); IR (thin film) 3400, 2930, 1600, 1500, 1450,1360, 1300, 1180, 1080 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.74 (d,J=6.6 Hz, 3H), 0.75 (s, 3H), 0.84 (d, J=7.1 Hz, 3H), 1.13 (m, 1H), 1.17(m, 1H), 1.23 (m, 1H), 1.35 (m, 1H), 1.4 (m, 2H), 1.53 (m, 1H), 1.6 (m,1H), 1.69 (m, 1H), 1.70 (s, 3H), 1.75 (m, 1H), 2.38 (s, 3H), 3.16 (br d,J=7.1 Hz, 2H), 3.62 (q, 2.4, 1H), 5.12 (br t, J=6.9 Hz, 1H), 6.23 (s,1H), 10.02 (s, 1H), 12.61 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ ppm 12.7(CH₃, C-12), 15.7 (CH₃, C-13), 16.1 (CH₃, C-15), 17.3 (CH₃, C-14), 21.5(CH₂, C-11), 23.8 (CH₃, C-22), 25.5 (CH₂, C-4), 32.4 (CH₂, C-8), 34.1(CH₂, C-3), 36.0 (CH, C-5), 36.1 (CH, C-7), 38.0 (C, C-6), 39.0 (CH,C-1), 70.4 (CH, C-2), 103.6 (C, C-18), 110.4 (CH, C-20), 112.0 (C,C-16), 121.8 (CH, C-10), 134.7 (C, C-9), 139.8 (C, C-19), 159.6 (C,C-17), 162.8 (C, C-21), 174.1 (C, C-23).

Phomopsichromin E (5)

Phomopsichromin E (5) was isolated as a white powder, C23H32O5; HRESIMSm/z 389.2363 [M+H]+, [α]²⁰D −0.4. By comparing 5 to 4 it was immediatelyevident that the only difference between the two was the increasedoxidation at C-2. Comparison to 1 and 4, along with the 2D data for 5(Table 5), completed the structure (FIG. 12).

TABLE 5 ID and 2D data for phomopsichromin E (5) in DMSO-d₆. Pos δ_(C)^(b) δ_(H) (m, J(Hz))^(a) HMBC^(c) 1 49.2 2.53 (q, 6.7, 1H) 2, 5, 6, 12,14 2 212.6 3 40.7 2.1 (m, 1H) 1, 2, 4, 5 2.4 (m, 1H) 4 30.3 1.47 (m, 1H)3, 5, 13 1.76 (m, 1H) 6 5 35.0 1.99 (m, 1H) 3, 4, 6, 13, 14 6 42.7 735.4 1.23 (m, 1H) 5, 6, 8, 14 1,32 (m, 1H) 1, 6, 8, 9, 14 8 32.0 1.8 (m,1H) 7, 9, 10, 15 1.91 (m, 1H) 7, 9, 10, 15 9 134.2 10 122.2 5,18 (br t,6.8, 1H) 8, 11, 15, 16 11 21.5 3.17 (d, 7.1, 2H) 9, 10, 16, 17, 21 127.5 0,76 (d, 6.6, 3H) 1, 2, 6, 7 13 14.7 0.81 (d, 6.6, 3H) 4, 5, 6, 1214 15.0 0.46 (s, 3H) 1, 5, 6 15 16.1 1.73 (s, 3H) 8, 9, 10, 16 16 111.917 159.6 —OH 12.62 (s, 1H) 18 103.5 19 139.9 20 110.4 6.23 (s, 1H) 16,17,18, 22 21 162. 8 —OH 10.04 (s, 1H) 16, 17, 20, 21 22 23.7 2.39 (s,3H) 18, 19, 20, 23 174.1 ^(a1)H NMR recorded at 600 MHz, reported in ppm(multiplicity, J-coupling in Hz, integration); ^(b13)C NMR recorded at200 MHz; ^(c)recorded from a gHMBCAD experiment at 600 MHz and reportedas positions of carbons.

Compound data for Phomopsichromin E (5) is as follows: C₂₃H₃₂O₅; HRESIMSm/z 371.2229 [M+H−H₂O]⁺ (C₂₃H₃₁O₄ calculated 371.2222), 389.2363 [M+H]⁺(C₂₃H₃₃O₅ calculated 389.2328), 411.2152 [M+Na]⁺ (C₂₃H₃₂O₅Na calculated411.2147); UV (MeOH) λmax (log ε) 220 (4.14); [α]²⁰D −0.4 (c 0.1, MeOH);IR (thin film) 3390, 2950, 1590, 1450, 1300, 1160, 1090, 1020 cm⁻¹; ¹HNMR Data (600 MHz, DMSO-d₆) δ ppm 0.46 (s, 3H), 0.76 (d, J=6.6 Hz, 3H),0.81 (d, J=6.6 Hz, 3H), 1.23 (m, 1H), 1.32 (m, 1H), 1.47 (m, 1H), 1.73(s, 3H), 1.76 (m, 1H), 1.80 (m, 1H), 1.91 (m, 1H), 1.99 (s, 1 H), 2.10(m, 1H), 2.39 (s, 3H), 2.40 (m, 1H), 2.53 (q, J=6.7 Hz, 1H), 3.17 (d,J=7.1 Hz, 2H), 5.18 (br t, J=6.7 Hz, 1H), 6.23 (s, 1H), 10.04 (s, 1H),12.62 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ ppm 7.5 (CH₃, C-12), 14.7(CH₃, C-13), 15.0 (CH₃, C-14), 16.1 (CH₃, C-15), 21.5 (CH₂, C-11), 23.7(CH₃, C-22), 30.3 (CH₂, C-4), 32.0 (CH₂, C-8), 35.0 (CH, C-5), 35.4(CH₂, C-7), 40.7 (CH₂, C-3), 42.7 (C, C-6), 49.2 (CH, C-1), 103.6 (C,C-18), 110.4 (CH, C-20), 111.9 (C, C-16), 122.2 (CH, C-10), 134.2 (C,C-9), 139.9 (C, C-19), 159.6 (C, C-17), 162.8 (C, C-21), 174.1 (COOH,C-23), 212.6 (C, C-2).

Bioactivities

As shown in Table 6 below, Phomopsichromins A (1), B (2), C (3), and E(5) all show reasonable activity against the infected macrophage modelof the Leishmania donovani parasite at 3, 1.9, 0.67, and 0.80 μM,respectively. Interestingly, neither phomopsichromin D (4) nor LL-Z1272ε(6) showed any activity towards the parasite or the macrophages.

Phomopsichromin C (3) was the only compound with any notable activityagainst any of the ESKAPE pathogens, with an MIC against MRSA of 58 μM.In a drug wash-out study, 3 was determined to be bactericidal.Impressively, this compound exhibits an MBC99 of 47 μM, which isreasonably close to the control gentamicin (20 μM in this assay).However, 3 also displays notable cytotoxicity towards HepG4 human livercells, with an LD50 of 37 μM. Further supporting its cytotoxicity andlack of specificity, 3 is also active against the macrophage containedLeishmania donovani as mentioned above.

1, 2, and 5 all displayed activity against MRSA at extremely highconcentrations (>500 μM) and further cytotoxicity testing may be useful.If these compounds continue to be selective against the Leishmaniadonovani parasite, further biological testing against that, and otherparasite targets would be warranted.

TABLE 6 Pure compound bioactivities of 1-6 Pure Compound BioactivitiesMRSA L. donovani Compound IC₅₀ (μM) IC₅₀ (μM) Other 1 518 3 2 515 1.9 358 0.67 MRSA MBC₉₉: 47 μM HepG4 LD₅₀: 37 μM 4 NA NA 5 515 0.80 6 NA 13

While none of the phomopsichromins immediately emerge as promising leadcompounds against ESKAPE or Leishmania donovani, with a group ofcompounds of this size, an interesting “natural structure activityrelationship (SAR) study” is beginning to take form. It is notable that4 remains inactive, despite being nothing more than the ring-open formof 2. Meanwhile, 1 and its ring opened form, 5, display similarbioactivities. While six compounds are hardly enough to constitute atrue SAR study, one can imagine that with the isolation (or synthesis)of more of these analogs, a pharmacophore may well begin to emerge. Afungal source is ideal for such studies as more material of these, andany other discovered compounds, could be obtained quite easily and inhigh quantities.

Materials and Methods

Microbial Isolation Protocols

Nutrient media components (SDB, PDB, TSA, Actinomycete Isolation Agar,Malt Extract Agar) and agar were produced by BD™ Difco™ and purchasedthrough Fisher Scientific. Glycerol, nystatin, cycloheximide, andchloramphenicol were purchased from Sigma Aldrich®. Solid media wasmixed, heated, and autoclaved according to manufacturer's instructionsand poured to set in Fisherbrand™ petri dishes.

After collections, field plates were incubated at 20-25° C. (roomtemperature), 4° C. (refrigerated), or 26-30° C. (heated) as sourceorganism or microbial targets dictated. Generally, bacterial plates wereheated and fungal plates were left at room temperature for collectionsin warm climates. Cold water microbial isolation generally took placewith refrigeration. Field plates were incubated for 1-4 weeks, anddisposed of after all colonies were isolated and/or after the wholeplate was covered in microbial growth. Isolated pure colonies were grownon SDA (fungi) and TSA (bacteria) for archiving as described above.Descriptions of each organism on this standard media were recorded.

Screening Protocols

Each fungal isolate was grown on SDA from either glycerol stocks orisolation plates, and after a colony was established, was subsampled forscreening. 1 cm cubes of fungal material and agar were inoculated intriplicate into 3 sterile Eppendorf™ tubes containing 1.25 mL each: SDB(control), 100 μM sodium butyrate in SDB (HDACi), and 100μM5-aza-cytidine in SDB (DNMTi). Sodium butyrate and 5-aza-cytidine werepurchased from Sigma Aldrich®. The SDB/modifier/fungal mixture wasagitated, and then each poured over 1 g rice media in a Fisherbrand™ 20mL glass scintillation vial. Rice media was made by autoclaving 1 g (¼tsp) of brown rice with 4 mL DI water. Once inoculated, rice vials wereincubated at 28° C. for 21 days. After 21 days, allcontaminated/non-growing culture sets were removed. Cultures werespritzed with ˜500 μL distilled MeOH. The fungal rice cake was brokenapart with a clean spatula. 10 mL distilled EtOAc was added using aglass 10 mL pipette and allow to extract overnight on bench top.Extracts were carefully decanted into clean, pre-weighed scintillationvials after 24 hours. Extracts were dried under air for 24 hours,resuspended in DMSO at a concentration of 10 mg/mL, and plated in96-well format on a TECAN Freedom EVO 150 liquid handling automatedworkstation. Five replicate Corning™ clear polystyrene 96-wellmicroplates were prepared with 150 μL of extracts/well for bioassay.Remaining extract material was stored in duplicate Fisherbrand™ 96-wellDeepWell™ polypropylene microplates. All extract plates were stored at−20° C.

LC-QToF-MS Protocols

Extracts were dried of DMSO and suspended in MeOH at a concentration of0.1 mg/mL. The resulting solution was filtered over 0.2 μm Phenomenex®RC syringe filters and were injected in triplicate on the LC-QToF-MS foranalysis.

Analysis was completed on an Agilent 6540 LC/QTOF with AgilentJet-stream Electrospray Ionization. A Kinetex C18 (5 μm, 100 Å, 2.1 mmID, 50 mm length) column was used.

Metabolomic and Statistical Analysis Protocols

Each sample for metabolomics analysis was prepared as above and run intriplicate according to the run parameters above. Resultingchromatograms were subjected to processing by Agilent MassHunterQualitative Analysis. First, a list of compounds was generated using theFind By Molecular Feature tool. A peak height cutoff was set at 100counts and results were limited to the largest 5000 compounds. Resultswere exported to .cef files that were transferred to Agilent MassProfiler Professional (MPP). In MPP replicates were averaged and blankswere subtracted. The resulting table of samples and chemical entities(mass @ retention time) was exported via Microsoft Excel and opened inPrimer 6 for statistical analysis (Clarke, K. R.; Gorley, R. N. 2006.PRIMER v6: User Manual/Tutorial. Primer-E, Plymouth). Abundances weresquare root normalized and factors such as culture conditions andbioactivities were added to each sample identity. A Bray-Curtissimilarity matrix was constructed from which cluster (dendogram) andmultidimensional scaling (MDS) plots could be created.

Scale-Up Protocols

A scale-up protocol that mirrored the screening culture protocol wasdeveloped. The optimized procedure was enacted for all scale-up levelscreening. Rice media was prepared in a Type 3T Unicorn bag according tothe following procedure: 300 g of brown rice was mixed with 500 mL DIwater and a heat sealer was used to seal the bag. Rice was autoclaved ona liquid cycle for 30 minutes at 121° C. Each hit organism chosen forscale-up was grown on SDA from either glycerol stocks or isolationplates, and after a colony was established, was subsampled forscreening. 1 cm cubes of fungal material and agar were inoculated intriplicate into 3 sterile 50 mL Falcon™ tubes containing 50 mL each: SDB(control), 100 μM sodium butyrate in SDB (HDACi), and 100μM5-aza-cytidine in SDB (DNMTi). Rice bags were cut open in a sterileenvironment, the fungal/liquid media mixture poured in, and resealed.Rice was agitated once a week for a 21 day culture period. Extractionwas completed by spritzing the culture with distilled MeOH to dampen thespores, transferring the material to a large beaker, and extractingovernight in a 1:3 MeOH:EtoAc mixture, followed by two 24-hourextractions in EtOAc. Extracts were collected, filtered, and dried downfor chemical analysis.

Conclusion

The Phomopsis sp. isolate proved to be a producer of new, bioactivechemistry and may have biosynthetic potential remaining to bediscovered. All six compounds discussed herein were isolated from theextract of the control growth condition. The extracts from the modifiedgrowth conditions remain to be explored. Additionally, NMR data suggeststhat there may be more phomopsichromins (or other known or newderivatives) as minor components of the investigated fractions that wereabandoned due to time constraints. This strain remains archived in thelab fungal library, along with the other 6 isolates from the branchpiece. These organisms are all known to produce bioactive extracts andwould all be interesting targets for further chemical analysis andgrowth culture optimization.

This genera is known to have great biosynthetic potential and despitebeing such a well-studied organism, environmental strains such as theone investigated here continue to be producers of new compounds.Environmental microbial isolates are a promising source of new chemistryfor drug discovery.

Fungal Library

General Protocol

Isolation techniques vary according to source and isolate targets, butcan be described by the following general protocol: 1) tissue surfacesterilization with a 10% bleach solution and/or isopropyl alcohol; 2)tissue subsampling into 1 cm cross-sections; 3) plating in triplicateonto solid media plates of variable composition; 4) careful monitoringof colony growth on solid media plates for a period of 1-4 weeks in thelab; and 5) transferring individual colonies to new isolation plates ofsimilar composition. The last step is repeated until a pure colony isestablished for each isolate.

Variations in solid media plate composition are employed to target awide range of fungi and bacteria, and all tissue samples collected areplated across 6-10 different media types to access the microbialdiversity that can be found living within each target organism.² Eachplate type is designed with the following general ingredients in varyingconcentrations: a nutrient source, agar, salt, and small moleculeantibacterial and antifungal agents in sub-lethal doses to discouragethe growth of fast-growing organisms. Available nutrient mixturesinclude: Sabaraud Dextrose Broth (SDB), Potato Dextrose Broth (PDB),Tryptic Soy Broth (TSB), Malt Broth, Actino Agar, and glycerol. Smallmolecule additives include: nystatin, cycloheximide, andchloramphenicol. Solid media types are curated for each collectionexpedition based on source and microbial targets.

Collection locations and source organisms represented in the isolatelibrary are varied. Some examples of organisms commonly sampled formicrobial isolation include: Floridian mangroves (Rhizophora mangle,Avicennia germinans, and Laguncularia racemose), mangrove associatedtrees (Conocarpus erectus, and Coccoloba uvifera), and benthic marineinvertebrates including sponges, tunicates, and corals. Collection date,location, and source organism identification is all carefully recordedin field notebooks during each expedition.

To accommodate different field conditions, two different collectiontechniques have been developed. The first technique is field plating, inwhich tissue samples are surface sterilized and directly plated onlocation onto solid media plates (“field plates”), which are thentransferred back to the lab for monitoring of growth. The secondtechnique is glycerol cryotube preservation, in which tissue samples aresurface sterilized, frozen and stored for transit in a 20% glycerolsolution in cryotubes before being plated in the lab. This allows formicrobial collections to take place around the world, preserving tissuesand micro-organisms until they can be processed in the lab.

After establishing a pure isolate, all micro-organisms are archived in a20% glycerol solution at −80° C. Bacterial cells are suspended in thesolution, while fungal material is stored as small cubes of growth cutfrom SDA isolation plates. Isolates are identified according to thefollowing nomenclature: “Collection Location, Year-Source OrganismNumber-Isolation Plate Type-Isolate Number”. In this way, isolates caneasily be grouped and retrieved according to any one of the collectionor isolation parameters. Nearly 75% of the existing fungal isolates inthis library were subjected to an epigenetics based high throughputscreening (HTS) project.

An epigenetics based fungal screening program was designed and a cultureminiaturization and modification protocol was developed to accommodatethe use of 20 mL scintillation vials and a brown rice media. The histonedeacetylase (HDAC) inhibitor sodium butyrate, and the DNAmethyltransferase (DNMT) inhibitor 5-azacytidine were employed atconcentrations of 100 μM for epigenetic modification. With a target ofscreening 500 organisms in 3 growth conditions (Control, HDACi, andDNMTi) each month for 12 months, goal-oriented timelines and protocolswere developed and put in place.

The resulting extract library was dispersed for screening and archivedin DMSO at a concentration of 10 mg/mL in 96-well plate format. Beyondthe scope of the original two grant funded screening targets (the ESKAPEpathogens and Leishmania donovani), extracts have been distributed forscreening in a number of additional targets, resulting in a body of datathat can inform high level prioritization of active extracts. Additionalscreening is ongoing, but extract data to date includes bioactivitiesagainst the ESKAPE pathogens, Leishmania donovani, J774 macrophagecells, Mycobacterium tuberculosis, Clostridium difficile, and Naegleriafowleri. The library was additionally screened against a number ofcell-based cancer targets and in a yeast based multiplex assay foranti-helminthics. Some active extracts were investigated by metabolomicanalysis.

Metabolic Analysis

Extracts from a subset of organisms producing bioactivities weresubjected to metabolomics analysis to investigate and confirm chemicaldiversity of the extract library and effectiveness of epigeneticmodification techniques. Bioassay results showed desirable distributionof active extracts among the three culture conditions, and in eachassay, organisms displaying activity only after modification accountedfor 15-30% of all activity observed (FIG. 13).

To support and further investigate this diversity of activity, 123extracts from 41 active organisms were analyzed via LC-QToF-MS. Thissubset of samples included active and non-active extracts (for instance,in the case that an organism only displayed activity after modification,the control extract was still included in the analysis). It included awide range of isolates from different collections, isolation media, andsource organisms. The LC-QToF-MS data were summarized into a list of‘chemical entities’ (HRESIMS @ retention time) for each sample usingAgilent Qualitative Analysis and Mass Profiler Professional software.Each sample, identified by its list of chemical entities, could then bestatistically compared using Multidimensional Scaling (MDS). Each samplewas also identified by a number of additional factors, includingbiological activity, culture treatment, and isolate identity. UsingPrimer 6 software, MDS plots were generated and annotated with any ofthese factors. This analysis allowed for a visual representation of thechemical similarity of the isolates and extracts to one another, andverified the chemical diversity of both the fungal library as well asthe different culture treatments.

It was found that some organisms share less than 20% similarity with theother analyzed organisms (e.g. KML12-14MG-B2a). Additionally, for someorganisms, different culture conditions induce an extract dissimilarityas high as 60% (e.g. EG10-47C-1). This data illustrates not onlychemical uniqueness between different fungal isolates, validating thecollection and isolation protocols, but also allows organisms in whichthe epigenetic modification has had a large impact on the metaboliteprofile to be identified. Organisms whose modified extracts exhibitunique chemistry and biological activities would be of high interest forscale up and chemical analysis. Through the processing for MDS analysis,this data is also ideally prepared for dereplication efforts. With arobustly annotated database of known cytotoxins, nuisance compoundscould be quickly identified and their extracts de-prioritized. This datarepresents an exercise in metabolomics analysis of a small subset ofscreened extracts, but could be scaled up and performed on the entireextract library for high-level analysis of all available chemistry.

Training Set Results

Another subset of the extract library (1305 extracts, 13% of the totallibrary) was screened fully against Mycobacterium tuberculosis (TB), theESAKPE pathogens, Leishmania donovani, Naegleria fowleri, and the J774murine macrophage cell line. Using stringent definitions of ‘active’ tomoderate the hit rate, 16% of these 1305 extracts were determined to beactive. While this number is quite large, this is a result of the factthat 77% of the hits were hits in only one assay. Within this subset,accounting for all bioactivities, the previously observed trends in eachof the individual assays (FIG. 13) were verified; the three culturetreatments were equally effective in producing active extracts, and 39%of active fungi only produced hits in the HDACi and DNMTi cultureconditions.

To analyze this data in the most meaningful way, strict activitycut-offs were set for each bioassay. For simplicity, the ESKAPEpathogens' MICs were reported as a single scaled score⁴ for all 6pathogens; an extract was considered ‘active’ in this assay if it had ascaled score≥7. For highest clinical relevance, only extracts exhibitingan IC50 value<1 μg mL-1 in the infected macrophage model of theLeishmania donovani parasite were included. TB activity was includedwhen ≥85%, and activity against Naegleria fowleri was defined asinhibition of >33% at either concentration tested (50 and 5 μg/mL). Anycytotoxicity against the J774 macrophage cells up to 20 μg/mL was alsoincluded. Results can be seen in FIG. 14.

Only 48 active extracts (23% of total active, 4% of total screened) werenot specific to a single target organism. 17 extracts (8%, 1%) wereactive only against N. fowleri, 53 (25%, 4%) against the ESKAPE panel,37 (17%, 3%) against Mycobacterium tuberculosis, 41 (20%, 3%) againstthe L. donovani infected macrophage, and 14 (7%, 1%) were cytotoxic onlyagainst the J774 macrophages. When the definition of ‘active’ wasrelaxed in a secondary hit, (i.e. an extract was only considered‘selective’ if it had reported activity only one of the 4 assays) 100extracts, 48% of active extracts, retained their qualification as‘selective’.

This is a unique set of data featuring extracts of diverse fungal originscreened against a wide range of eukaryotic and prokaryotic diseasecausing organisms. The specificity of bioactivity in this data setsuggests unique underlying chemical profiles and, gratifyingly, providesstrong support to the screening program design. With this data, theinventors have demonstrated that vigorous front-end investigation(multiple bioassays, metabolomic analyses, dereplication, etc.) of alibrary of extracts can inform scale-up prioritization in a highlyeffective way for the greatest chance of isolating new, bioactivenatural products. Scale-up efforts are underway to further validatethese methods.

With such promising data from the extract library, scale-ups ofbioactive fungi have commenced. A scale-up protocol was developed andsamples prioritized according to data available at the time. With thecompleted analysis of the training set, newly prioritized organisms havebeen identified and are available for chemical investigation in future.Extracts that feature specific, potent, and induced bioactivities can bescaled up and subjected to chemical analysis including new dereplicationprotocols as updated databases become available. With the plethora offront-end analysis that has been employed and presented here, organismscan now be chosen for chemical investigation with confidence in thechances of discovering new, bioactive secondary metabolites.

New Citreohybriddione

Citreohybridones and Citreohybriddiones

The hybrid strain KO 0031 of Penicillium citreo-viride B. IFO 6200 and4692 has been reported to be a prolific producer of meroterpenoidsecondary metabolites. (Kosemura, S. Meroterpenoids from Penicilliumcitreo-viride B. IFO 4692 and 6200 Hybrid. Tetrahedron 2003, 59 (27),5055-5072). Many of these hybrid polyketide-terpenoids are known to befeeding deterrents against the crop pest Plutella xylostella. (Kosemura,S.; Yamamura, S. Isolation and Biosynthetic Pathway for Citreohybridonesfrom the Hybrid Strain KO 0031 Derived from Penicillium species.Tetrahedron Lett. 1997, 38 (35), 6221-6224; Kosemura, S.; Matsunaga, K.;Yamamura, S. Citreohybriddiones A and B and Related Terpenoids, NewMetabolites of a Hybrid Strain KO 0031 Derived from Penicilliumcitreo-viride B. IFO 6200 and 4692. Chem. Lett. 1991, 1811-1814;Kosemura, S.; Matsou, S.; Yamamura, S. Citreohybriddione C, AMeroterpenoid of a Hybrid Strain KO 0031 Derived from Penicilliumcitreo-viride B. IFO 6200 and 4692. Phytochemistry 1996, 43 (6),1231-1234; Kosemura, S.; Miyata, H.; Yamamura, S.; Albone, K.; Simpson,T. J. Biosynthetic Studies on Citreohybridones, Metabolites of a HybridStrain KO 0031 Derived from Penicillium citreo-viride B. IFO 6200 and4692. J. Chem. Soc. Perkin Trans 1994, No. 1, 135-139). The previouslyreported citreohybridones and citreohybriddiones are two groups of thesefungal natural products (FIG. 15). The inventors have isolated a newcitreohybriddione (citreohybriddione D, 1) from an environmentalPenicillium sp. fungus.

KML12-14MG-B2a Isolation

A number of collection trips to the Keys Marine Lab, Long Key, Fla. havetargeted mangrove endophytes. Traveling by canoe and using field platingtechniques, samples were collected from areas all around the facilitytargeting a broad range of micro-organisms. The Keys Marine Lab isuniquely located with access to a wide variety of mangrove environments.

KML12-14MG-B2a is a Penicillium sp. isolated from the 2012 Keys MarineLab trip. This organism was isolated from the stem tissue of a juvenileRhizophora mangle tree in an exposed mangrove community on the west sideof Long Key. This fungus was isolated on a solid water agar platecontaining sub-lethal doses of both nystatin and cycloheximide. On SDAthis organism has a typical Penicillium morphology, growing radiallyfrom the inoculated mycelia with a light green/white color and fluffysporulating bodies.

Initial Bioactivities, Scale-Up, Epigenetic Modification, andIdentification

Similar to other fungal isolates in the microbial library,KML12-14MG-B2a was cultured and screened as a part of the screeningprogram discussed above. This isolate emerged early as an important hitagainst the ESKAPE pathogens, hitting against both gram positive andgram negative pathogens in the Control and HDACi growth conditions(Table 7). In support of the design of the screening program, the HDACiculture produced a consistently active extract across replicatecultures, while repeated Control cultures produced inconsistentactivity. Demonstrating the ability of these organisms to regulate theirbiosynthetic machinery and the effectiveness of the HDACi cultureconditions, KML12-14MG-B2a became a model organism for scale-up andchemical investigation protocols.

TABLE 7 Screening results for KML12-14MG-B2a. Sample Information MIC(μg/mL) Date Loc Extract ID 200 100 50 25 10 Jan. 9, 2014 F1KML12-14MG-B2a (HIT) CONTROL EKAP EKAP EKAP EAP EA Jan. 9, 2014 F2KML12-14MG-B2a (HIT) HDAC EKAP EKAP — — — Jan. 9, 2014 F3 KML12-14MG-B2a(HIT) DNMT — — — — — Feb. 3, 2014 P2 D7 KML12-14MG-B2a (HIT) CONTROL — —— — — Feb. 3, 2014 P2 D8 KML12-14MG-B2a (HIT) HDAC ESKAP ESKAP ESKAP EKPEK Feb. 3, 2014 P2 D9 KML12-14MG-B2a (HIT) DNMT — — — — — This organismwas screened multiple times as a part of a subset of isolates chosen toverify reproductibility in the screening protocol. Activity is indicatedby the first letter of the pathogen the extract showed activity against.E = Enterococcus faecium, S = Staphylococcus aureus, K = Klebsiellapneumoniae, A = Acinetobacter baumannii, P = Pseudomonas aeruginosa.

KML12-14MG-B2a was scaled up in control culture conditions on 700 g ofbrown rice (autoclaved with 1.4 L DI water) and inoculated in 100 mL ofSDB in 2 types of Unicorn brand mycobags and a 3 L fernbach flask. After21 days of incubation, both types of mycobag (Unicorn types 3T and 14A)were found to produce sterile, fully optimized growth (i.e. all ricematerial covered with growth) that resulted in extracts of similar mass.The fernbach flask culture proved significantly less desirable, withfungal growth only on the top of the solid rice due to an inability toagitate the culture throughout the culture process.

Epigenetically modified cultures on KML12-14MG-B2a were then scaled upusing the same protocols (at this time, Unicorn bag types 3T and 14Awere used interchangeably according to supply. Type 3T bags were laterpurchased in bulk and all further scale-ups done in those bags). Again,resulting growth was uncontaminated and represented complete mediacoverage. The cultures each resulted in approximately 70 g of crudeextract after a 3 day exhaustive extraction (day 1: 50 mL MeOH, 750 mLEtOAc; days 2 and 3: 800 mL EtOAc). Extracts were filtered over celiteand sent for bioassay. Unfortunately, the crude extracts of thescale-ups did not replicate the activity seen in the small scalecultures. A liquid:liquid partition between EtOAc and H₂O, followed byNP MPLC separation of the EtOAc partition was performed on each extract,and the resulting fractions sent for bioassay. Again, disappointingly,the fractions returned inactive. Scale-up optimization efforts werecontinued with other organisms in search of a scale up protocol thatcould be standardized and used in a scale-up screening program. Due tointeresting NMR data, chemical investigation continued on the fractionsof the HDACi extract of KML12-14MG-B2a.

KML12-14MG-B2a was identified using sanger sequencing of the 18Sribosomal spacer region. Agreeing with the previously observedmorphology, the isolate was identified to the genus level as aPenicillium sp.

Compound Isolation and Structure Elucidation

The MPLC separation of KML12-14MG-B2a HDACi resulted in 10 fraction(FIG. 16). With no bioactivity in any of the fractions, compoundelucidation proceeded via NMR guided fractionation. NMR analysis of all10 fractions identified fraction F as interesting for furtherinvestigation. HPLC separation of F was completed in normal phase(n-hexanes:ethyl acetate) on a silica column to yield 10 fractions (FIG.17). Fraction F-2 was discovered to contain the new meroterpenoid,citreohybriddione D (1).

Fraction F-2 was further purified on silica to yield compound 1. The ¹HNMR spectrum of 1 was notable in that it contained 8 methyl signals(Table 8) and almost nothing in the olefin region. Investigation of the¹³C data confirmed the presence of a large number of quaternary carbons,indicating a highly functionalized fused ring system such as apolyketide. The HRESIMS of 503.2640 [M+H]⁺ resembled the terpenoidskeletons of the citreohybridones and citreohybriddiones, but did notmatch any of the previously reported compounds. ¹³C NMR signals ofcarbons 1-12 strongly matched citreohybridone D, however carbons 13-18more closely resembled citreohybriddione B.

TABLE 8 1D and 2D data for citreohybriddione D (1) in CDCl₃. Pos δ_(C)^(b) δ_(H) (m_(,) J(HZ))^(a) HMBC^(c) 1 27.8 1.03 (m, 1 H) 2, 9, 10, 22,23, 25 2.38 (m, 1 H) 3, 5, 9, 10 2 23.3 1.6 (m, 2 H) 10 3 76.8 4.68 (t,2.6, 1 H) 1, 5, 26 4 36.9 5 47.7 1.83 (m, 1 H) 4, 6, 10, 23, 24 6 16.91.8 (m, 1 H) 4, 8, 10 2.01 (m, 1 H) 7, 10 7 30.8 2.83 (m, 1 H) 5, 6, 8,22 2.38 (m, 1 H) 5, 9, 10 8 38.6 8, 10, 11, 12, 23 9 53.5 2.21 (m, 1 H)10 52.2 11 126.4 5.85 (s, 1 H) 8, 9, 10, 13, 21 12 133.0 13 60.8 14 70.415 210.6 16 72.1 17 206.7 18 19.8 1.4 (s, 1 H) 15, 16, 17 19 167.3 2016.4 1.31 (s, 3 H) 12, 13, 14, 17 21 18.8 1.7 (s, 3 H) 11, 12, 13 22 1951.17 (s, 3 H) 7, 8, 9, 14 23 204.4 10.14 (s, 3 H) 1, 10, 22 24 26.5 0.97(s, 3 H) 2, 3, 4, 5, 25 25 1.3 0.9 (s, 3 H) 3, 4, 5, 24 3-OAc 170.63-OAc 21.3 2.12 (s, 3 H) 26 16-OH 2.16 (s, 1 H) 15, 16, 17 19-OMe 52.03.63 (s, 3 H) 19 ^(a1)H NMR recorded at 500 MHz, reported in ppm(multiplicity, J-coupling in Hz, integration); ^(b13)C NMR recorded at200 MHz; ^(c)recorded from a gHMBCAD experiment at 500 MHz and reportedas positions of carbons.

With conformation from 2D NMR experiments (FIG. 18), it was determinedthat 1 is, in fact a new citreohybriddione lacking any lactone orepoxide ring structures. 2D NOESY data confirmed that the absolutestereochemistry matches what has been previously reported for similarcompounds (FIG. 19). (Kosemura, S. Meroterpenoids from Penicilliumcitreo-viride B. IFO 4692 and 6200 Hybrid. Tetrahedron 2003, 59 (27),5055-5072).

NOE correlations between the methoxy at C-19 (δ_(H) 3.63) and the methylgroups at C-18 (δ_(H)1.40) and C-20 (δ_(H) 1.31) confirmed that theD-ring in 1 is up as in the other citreohybriddiones. The alcohol onC-16 of 1 has the same orientation as that in citreohybriddione B.Through-space correlations between C-22, 23, and 25 confirm that thestereochemistry of the A and B rings of 1 are as reported incitreohybriddione A.

Compound data for Citreohybriddione D (1) as follows: C₂₈H₃₈O₈; HRESIMSm/z 443.2431 [M−OAc]⁺ (C₂₆H₃₅O₆ calculated, 443.2434), 503.2640 [M+H]⁺(C₂₈H₃₉O₈ calculated, 503.2645), 525.2465 [M+Na]⁺ (C₂₈H₃₈O₈Nacalculated, 525.2464); [α]²⁰ _(D) +0.4 (c 0.1, MeOH); ¹H NMR Data (500MHz, CDCl₃) δ ppm 0.90 (s, 3H), 0.97 (s, 3H), 1.03 (m, 1H), 1.17 (s,3H), 1.31 (s, 3H), 1.40 (s, 3H), 1.68 (m, 1H), 1.70 (m, 3H), 1.80 (m,1H), 1.83 (m, 1H), 2.01 (m, 1H), 2.12 (s, 3H), 2.16 (s, 1H), 2.21 (m,1H), 2.38 (m, 1H), 2.83 (m, 1H), 3.63 (s, 3H), 4.68 (t, J=2.6 Hz, 1H),5.85 (s, 1H), 10.14 (s, 1H); ¹³C NMR (200 MHz, CDCl₃) δ ppm 16.4 (CH₃,C-20), 16.9 (CH₂, C-6), 18.8 (CH₃, C-21), 19.5 (CH₃, C-22), 19.8 (CH₃,C-18), 21.3 (CH₃, C-25), 21.3 (CH₃, C-27), 23.3 (CH₂, C-2), 26.5 (CH₃,C-24), 27.8 (CH₂, C-1), 30.8 (CH₂, C-7), 36.9 (C, C-4), 38.6 (C, C-8),47.7 (CH, C-5), 52.0 (CH₃, C-28), 52.2 (C, C-10), 53.5 (CH, C-9), 60.8(C, C-13), 70.4 (C, C-14), 72.1 (C, C-16), 76.8 (CH, C-3), 133.0 (C,C-12), 126.4 (CH, C-11), 167.3 (C, C-19), 170.6 (C, C-26), 204.4 (CH,C-23), 206.7 (C, C-17), 210.6 (C, C-15).

Conclusions

Citreohybriddione D (1) was isolated from the HDACi treatment extract.Initial investigation via HPLC of similar fractions indicated that 1 mayhave also been present in the DNMTi extract as well, but appeared absentin the control treatment fractions. However, HRESIMS investigation ofthe ethyl acetate partitions of all three treatment extracts revealedthat 1 could be found in all treatments. Nevertheless, HRESIMS and NMRanalysis shows that there are notable chemical differences between thecontrol and modified conditions, indicating that there may be many othernew compounds to be found from this Penicillium sp. yet.

The culture optimization of KML12-14MG-B2a was not completed due to timerestraints. This organism remains active in the small scale, andwarrants further study to be able to replicate that activity on aculture scale that allows for chemical investigation of the activecompound(s). The isolation of 1 confirms that this Penicillium sp.contains PKSs (polyketide synthases) that are capable of producinginteresting secondary metabolites. Additionally, the small scalescreening results confirm that this organism responds favorably to HDACitreatment, indicating that efforts in culture optimization would likelybe rewarded with the production of many otherwise silenced naturalproducts.

While there are no reported activities for any of the citreohybriddiones(including 1) against human disease, further testing of this newcompound is warranted. Due to mass limitations, 1 was only screenedagainst the ESKAPE pathogens and the Leishmania donovani parasite.Feeding studies and cytotoxicity profiling are needed to determine howit compares to the rest of the compounds in this class.

In the preceding specification, all documents, acts, or informationdisclosed does not constitute an admission that the document, act, orinformation of any combination thereof was publicly available, known tothe public, part of the general knowledge in the art, or was known to berelevant to solve any problem at the time of priority.

The disclosures of all publications cited above are expresslyincorporated herein by reference, each in its entirety, to the sameextent as if each were incorporated by reference individually.Furthermore, where a definition or use of a term in a reference, whichis incorporated by reference herein, is inconsistent or contrary to thedefinition of that term provided herein, the definition of that termprovided herein applies and the definition of that term in the referencedoes not apply.

The advantages set forth above, and those made apparent from theforegoing description, are efficiently attained. Since certain changesmay be made in the above construction without departing from the scopeof the invention, it is intended that all matters contained in theforegoing description or shown in the accompanying drawings shall beinterpreted as illustrative and not in a limiting sense.

While there has been described and illustrated specific embodiments ofthe invention, it will be apparent to those skilled in the art thatvariations and modifications are possible without deviating from thebroad spirit and principle of the present invention. It is also to beunderstood that the following claims are intended to cover all of thegeneric and specific features of the invention herein described, and allstatements of the scope of the invention which, as a matter of language,might be said to fall therebetween.

What is claimed is:
 1. A method of inhibiting microbial activity in at least one cell comprising: contacting the at least one cell with an effective amount of a Citreohybriddione compound, wherein the Citreohybriddione compound has the following formula:


2. The method of claim 1, wherein the microbial activity is caused by Leishmania donovani, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, or Enterobacter cloacae.
 3. The method of claim 1, wherein the microbial activity is caused by Enterococcus faecium, Klebsiella pneumoniae, Acinetobacter baumannii, or Pseudomonas aeruginosa. 